| Background:MicroRNAs (miRNAs) are a relatively novel class of endogenous, non-protein coding small RNA molecules that can regulate hundreds of genes through binding to the3’UTR of target mRNAs. MiRNAs have been proven to play important roles in self-renewal, cellular development, differentiation, proliferation and apoptosis. Mature miRNAs bind to specific regions of mRNA targets by a mechanism that is not completely understood, resulting in translational repression or mRNA degradation. The human genome encodes more than1,000miRNAs with tissue or cell type specific expressions. Studies have shown that miRNAs have been observed in a wide range of human diseases, including autoimmune, inflammatory, neurodegenerative and cardiovascular diseases.There are mounting evidences showing that cardiac-specific miRNAs, miR-1, miR-133a/b and miR-208, are involved in heart development and cardiovascular diseases, including acute myocardial infarction (AMI) in experimental animals. Recent studies have shown that some miRNAs are present in the systemic circulation, both in humans and animals, and are associated with exosomes and microparticles. The levels of some circulating miRNAs have been reported to be differentially expressed during pregnancy and in the presence of a variety of cancers or cardiovascular diseases.High mortality and morbidity is common in AMI. Some biomarkers have been applied in diagnosis of AMI; for example, cardiac troponin I (cTnI), MB and so on. New approaches that can improve current diagnosis for AMI are still lacking. However, the expression and possible roles of miRNAs in AMI are less well studied. Cardiac injury, which occurs after AMI increases the circulating levels of several myocardial-derived miRs (eg, miR-1, miR-133, miR-499, miR-208). miRNAs in cardiovascular system are supported by the findings that depletion of the miRNA-processing enzyme Dicer lead to defects in vessel formation, angiogenesis and cardiac development.Acute myocardial infarction (AMI) is one of the most serious cardiovascular diseases. An early and accurate diagnosis can guarantee immediate initiation of reperfusion therapy to potentially reduce the mortality rate. Recent studies suggested that circulating myocardial-derived miRNAs might be useful aspotential biomarkers for infarction.Previous studies demonstrated that miR-30a was associated with hypertrophy and that miR-195was up-regulated during cardiac hypertrophy in mice. Validated targets of miR-195regulated apoptosis, proliferation and cell cycle. Moreover, recent studies showed a pro-apoptotic role of miR-195in cardiomyocytes. It was shown that expression of let-7was down-regulated in myocardial-injury mode. Meanwhile, thioredoxin1induced over-expression of let-7family inhibited cardiac hypertrophy. Recently, it was reported that AMI modulated miR-1, miR-133a/b, and miR-499-5p plasma levels in humans and mice. These results suggested that miRNAs may have fundamental roles in myocardial diseases. However, the expression levels of circulating miR-30a, miR-195and let-7b in AMI remained unknown. In this study, we assessed the hypothesis that circulating miR-30a, miR-195and let-7b may be useful for identifying and evaluating AMI.In the present study, we assessed the hypothesis that miR-30a, miR-195, let-7b, microRNA-1and microRNA-126might be useful for identifying and evaluating AMI. Our aim was to look for miRNA-based modulation of gene expression in AMI.Methods:(1) After obtaining the written informed consents,5ml blood amples were obtained from18patients and30healthy adults at Tongji Hospital from October2009 to May2010.(2) The blood samples of patients with AMI were obtained at4h (±30min),8h (±30min),12h (±30min),24h (±30min),48h (±30min),72h (±30min) and1w (±60min) after the onset of symptoms. Plasma was isolated by centrifugation and was maintained at-80±until RNA extraction.(3) Plasma cTnI concentrations were measured by ELISA assay according to the manufacturer’s protocol.(4) Total RNA was extracted from plasma using TRIzol LS Reagent.(5) The relative expression levels for each miRNA were calculated by the comparative CT method. To avoid possible differences in the amount of starting RNA, resultant miRNA levels were normalized to small nuclear RNA U6.(6) All statistical analyses were performed using the statistical software SPSS13.0(Statistical Package for the Social Sciences, Chicago,Ill) for Windows. Relative miRNA expression level was calculated by2-△△ct method. Independent-samples T test was used for two-group comparisons. Comparisons of parameters among≧3groups were analyzed by repeated measures ANOVA. For categorical variables, the Chi-Square test was used. MiRNAs and cTnI time course trends were analyzed by repeated-measures ANOVA. All tests were2-sided and a significance level of P<0.05(95%CI) was considered statistically significant. The ability to distinguish AMI group from control group was characterized by the receiver operating characteristic (ROC) curve, and the area under the ROC curve (AUC) was calculated.Results:(1) Using qRT-PCR, we analyzed the expression levels of three miRNAs (miR-30a, miR-195and let-7b) in AMI patients and healthy adults. Plasma miR-30a in patients with AMI exhibited a1.4(60.37) fold,10.48(62.75) fold and1.45(60.34) fold increase at4h,8h and12h, respectively. Similarly, miR-195exhibited a10.2(61.61) fold and1.4(60.3) fold increase in AMI group compared with control group at8h and12h, respectively. Interestingly, both miR-30a and miR-195reached their circulating expression peak at8h compared with other time points.(2) miR-1, miR-126and cTnI time courses exhibited the same trends in these patients, by repeated-measures ANOVA analysis. Importantly, no significant interaction effects were found in the time course between miRNAs and cTnl.(3) Differently from the individual miRNA-score, the composite-miRNA-score represented the cumulative plasma levels of the three miRNAs (miR-30a, miR-195and let-7b) with a strong differentiation for the comparison between AMI and controls, which was described in the methods section. The median score of composite-miRNAscore were2.93and2.96in AMI group and1.53and1.56in control group at8h and12h, respectively. The ability of the composite-miRNA-score to distinguish AMI group from control group was showed by the ROC curve with an AUC of0.93and0.92. By using a threshold score of1.815and2.025, above which patients were predicted to belong to the AMI group, a sensitivity of94%and90%, and a specificity of90%and90%were achieved for the identification of AMI patients, respectively.(4) The miR-1and miR-126-scores allowed a significant separation between the AMI and control groups (P=1e-7). The ability of the miR-1-score to differentiate the AMI group from the control group is demonstrated by the ROC curve with an AUC of0.92,0.90,0.94,0.92,0.96and0.90at4h,8h,12h,24h,48h and72h (Figure3and Table2). We achieved a sensitivity of93%,93%,94%,93%,93%and90%and a specificity of90%,90%,93%,90%,90%and90%, respectively, for the identification of AMI patients. The ROC curve of miR-126showed moderate ability to distinguish between the AMI group and the healthy control group at4h,8h,12h,24h,48h and72h with an AUC of0.86,0.87,0.88,0.87,0.84and0.83(Figure5and Table2), respectively. We gained a specificity of78%,84%,88%,77%,80%and70%and a sensitivity of81%,81%,81%,81%,81%and81%, respectively, for the diagnosis of AMI in patients.Conclusion:Our results imply that the plasma concentration of miR-30a, miR-195, let-7b, miR-1and miR-126can be potential indicators for AMI. Background:Stroke is a leading cause of death and long-term disability in developed countries, and-80%of strokes are ischemic in origin. In China,2.5million people have stroke and1million die from stroke-related causes every year. Multiple risk factors for stroke include advanced age, diabetes mellitus, hypercholesterolemia, hypertension, alcohol, smoking et al.. MicroRNAs (miRNAs) are a novel family of nonprotein-coding short RNA molecules that regulate gene expression brecognizing binding sites located in the3’untranslated region (3’UTR) of mRNA targets. MiRNAs participate in a large number of physiological and pathological processes, such as differentiation, development, proliferation, apoptosis and migration.However, compared with oncology or cardiology researches, a few studies have investigated the roles of miRNAs in neuronal death, degeneration or ischemic stroke. For instance, progressive neurodege neration occurs in the absence of Dicer, which is the crucial regulat or of miRNA biogenes and miR-8targets atrophin to prevent neurode generation in Drosophila.The miR-146A/G allele was found to be associated with ischemic stroke pathogenesis. MicroRNA-195protects against dementia induced by chronic brain hypoperfusion viaits anti-amyloidogenic effect in rats. The involvement of miRNA in regulating the pathogenesis associated with middle cerebral artery occlusion (MCAo) in SD rats was first reported by Jeya seelan et al., which demon strated that miR-30a-3p was down-regulated in the24-hour-reperfused MCAo rat brains but was subsequently up-regulated during the48-hour reperfusion. Recent studies indicate that miR-30family regulates angiogenesis, and endothelium specific miRNA-miR-126was down-regulated in young stroke patients. Moreover, the expression of LIN28B and let-7miRNA correlated with rs17065417in neuroblastoma cell lines. Let-7activates Toll-like receptor7that contributes to the spread of CNS damage.Acute myocardial ischemia and ischemic stroke have similar pathophysiology, and our previous studies implied that the plasma concentration of miRNAs can be potential indicators of AMI. Using the levels of circulating miR-30a, miR-126and let-7b at early phase of AMI, we were able to define a score with a high sensitivity and specificity for the detection of AMI patients.However, it is not clear whether miR-30a, miR-126and let-7b are involved in ischemic stroke and specifically, assosiation of their plasma levels and ischemic stroke has not been reported. In the present study, we assessed the hypothesis that circulating miR-30a, miR-126and let-7b might be useful for identifying and evaluating ischemic stroke in humans.Methods:(1) Blood samples Experiments were conduted in accordance with theprinciples of Declaration of Helsinki. This study was approved by the Ethics Committee of Tongji Hospital. Written informed consents were obtained from all the participants and247blood samples (5ml) were collected from the ischemic strok e patients and healthy volunteers at Tongji hospital from June2009to October2009. The study included first-ever stroke patients with cerebral infarction. Diagnosis was based on the International Classification of Diseases, Ninth Revision as described previously. Imaging studies were reviewed by experienced neuroradiologists to confirm the diagnosis and identify the stroke subtypes.The ischemic stroke patients identified by World Health Organization clinical criteria were further classified according to TOAST classification, a) large-vessel atherosclerosis (LA, n=51); b) small-vessel disease (SA, n=48); c) cardioembolism (CEmb, n=50); d) undetermined cause (UDN, n=48). The patients’functional status at the time of blood sampling was evaluated with the modified Rankin Scale (mRS). This study was a cross-sectional study, stroke patients were recruited at different time points after stoke, and each time point was represented by a different set of patients. The blood samples of patients with ischemic stroke were obtained at24h (with in24h),1w (±24h),4w (±24h),24w (±48h) and48w (±72h) after the onset of symptoms. Plasma was isolated by centrifugation and was maintained at-80℃until purification.(2) The relative expression level of each miRNA was calculated using the comparative CT method. MiRNA expression was normalized to small nucleolar RNA U6. Relative expression of miRNA was calculated using2"△△ct method in duplicate experiments. MicroRNA expression was normalized to endogenous control U6. All value s of miRNAs are expressed as mean±SD. For categorical variables, the Chi-Square test was used. Independent samples t-test was used for2-group comparisons. Differences were defined as statistically significant at a value of P<0.05. A composite score (denoted as miRNA-score) was defined to represent the cumulative levels of the miRNA (miR-LA, miR-SA, miR-CEmb and miR-UDN) in the ischemic stroke group compared with the control group as described previously. The miRNA-score of each sample was calculated as the sum of the inverted-normalized signals of the miRNA and adjusted by subtracting a constant (the minimal score) so that the range of scores starts at0. The ability to distinguish the ischemic stroke group and control group was characterized by the receiver operating characteristic (ROC) curve, and the area under the ROC curve (AUC) was calculated. All statistical calculations were performed using SPSS13.0for Windows.Results:(1) MiRNAs plasma levels in ischemic stroke patients and healthy volunteers. Using qRT-PCR assays, we measured the circulating levels of miR-30a, miR-126and let-7b in ischemic stroke patients and healthy controls. There were no significant differences among plasma miRNAs collected from patients and control at48w but it was found that circulating miR-30a and miR-126were down-regulated in ischemic stroke patients at24h,1w,4w and24w. Plasma levels of miR-30a in all subtypes of ischemic stroke patients’ were45%-79%lower than the controls at24h,1w,4w and24w. Plasma levels of miR-126subtypes of ischemic stroke patients were85%-98%lower than the healthy controls at24h,1w,4w and24w. Interestingly, our data showed that the expression pattern of circulating let-7b in ischemic stroke patients with large-vessel atherosclerosis were different from patients with other subtypes. Specifically, plasma levels of let-7b from ischemic stroke patients with large-vessel atherosclerosis (let-7b-LA) were70%-75%lower than in heathy controls at24h, lw,4w and24w. However, it was hihly expressed in other subtypes of ischemic stroke patients (let-7b-SA, let-7b-CEmb and let-7b-UDN) at24h,1w,4w and24w exhibiting3.51-14.42fold increase. Moreover, circulating levels of these miRNAs were also detected in blood samples from hemorrhagic stroke patients. The results showed that the circulating levels of the three miRNAs did not change at the different time points examined.(2) Since the expressions of miRNAs may be affected by both technical and biological variation, we combined the levels of each miRNAs from different subtypes at the same time point into a single score to increase the signal to noise ratio. A miR-score represents the cumulative level of the miRNA (miR-L A, miR-SA, miR-CEmb and miR-UDN) for the comparison between ischemic stroke group and control group, which was described in the methods section. The miR-scores distinguished the ischemic stroke patients and the controls clearly. The ability of the miR-30a-score to differentiate the stroke group from the control group was revealed further by the ROC curve with an AUC of0.91(95%CI=0.869-0.979),0.91(95%CI=0.848-0.971),0.92(95%CI=0.856-0.976) and0.93(95%CI=0.875-0.984) at24h,1w,4w and24w, respectively. We obtained a sensitivity of94%,93%,90%and92%and a specificity of80%,84%,84%and84%at24h,1w,4w and24w, respectively. And the ROC curves of miR-126with an AUC were0.92(95%CI=0.871-0.978),0.94(95%CI=0.895-0.985),0.93(95%CI=0.878-0.982) and0.92(95%CI=0.864-0.977). The sensitivity of miR-126-score for the diagnosis of stroke was92%,90%,92%and92%, and the specificity was84%,86%,84%and82%at24h,1w,4w and24w, respectively. Finally, the ability of the let-7b-score to distinguish stroke group from control group was shown by the ROC curve with an AUC of0.93(95CI=0.879-0.980),0.92(95%CI=0.866-0.98),0.92(95%CI=0.858-0.98) and0.91(95%CI=0.849-0.97). We achieved a sensitivity of92%,90%,92%and89%, and a specificity of84%,84%,86%and80%for identification of ischemic stroke patients at the above mentioned time points.Conclusions:In summary, our data demonstrated a significant change in the circulating levels of miR-30a, miR-126and let-7b in patients with ischemic stroke suggesting that miR-30a, miR-126and let-7b might be the potential biomarkers for the diagnosis of ischemic stroke. |
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